This chapter describes the preparation of primary human epidermal keratinocyte cultures and some assays which have been used to assess cellular viability or cytotoxicity following chemical exposure. It discusses in vitro cell or tissue culture approaches to the assessment of dermal irritation. The development of valid biochemical, cellular, and tissue based in vitro dermal irritation/sensitization models should generate additional information, which will help establish the predictiveness of structure-activity models. Animal models used to assess dermal irritancy, sensitization, and corrosion are accepted as valid due to their ability to serve as a basis to predict human response and because dermal inflammation, immunoresponse, and ulceration involve complex processes which are highly integrated and regulated in vivo. No in vitro alternative assay to predict chemically induced dermal inflammation has been formally validated and proven acceptable for the replacement of any in vivo dermal inflammatory test.

Abstract : The mouse ear model has been used at the U.S. Army Medical Institute of chemical Defense (USAMRICD) to assess dermal injury caused by sulfur mustard (HD) and to evaluate potential pre- and posttreatment compounds for efficacy against percutaneous HD injury. In this model, the effects of HD with or without treatment compounds are evaluated macroscopically by edema and microscopically by histopathological changes associated with HD injury. The objectives of Task 9543 were first to transition this technique to the Battelle Medical Research and Evaluation Facility (MREF), and then to evaluate candidate antivesicant drugs for their efficacy at eliminating or lessening the effects of HD-induced tissue damage. By June 1997 the mouse ear model had been transitioned and validated at the MREF. The MREF has evaluated 334 candidate HD treatment compounds provided by USAMRICD, and 73 passed the initial screening. Of these, 23 were anti-inflammatory agents, 21 were protease inhibitors, 15 were inhibitors of poly-ADP-ribose polymerase, 12 were HD scavengers, and 2 were other classes of compounds. Several of the successful HD scavengers and anti-inflammatory agents were also tested as posttreatments (applied 10 minutes after HD) and showed some efficacy.

: The objective of this study was to investigate the pharmacologic modulation of biomarkers for evaluating therapeutics against HD. The molecular biomarkers investigated, using a ribonuclease protection assay (RPA) to determine messenger ribonucleic acid (mRNA) levels, were mediators of inflammation, including several cytokines and chemokines, tenascin, and ornithine decarboxylase. Biochemical biomarkers investigated were serum amyloid P (SAP), interleukin-6 (IL-6), and interleukin-1 alpha (IL-1alpha) using ELISAs, and activity of myeloperoxidase (MPX) using a spectrophotometric method. Other endpoints included histopathology and edema measurements. Exposure to HE) resulted in a time-dependent increase in mRNA levels of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MiP-2), macrophage inflammatory protein-1 alpha (MIP-1alpha) and interleukin- 1 beta (IL-1Beta). Exposure to HD also resulted in edema and apparent increases in the protein levels of SAP and IL-6, and in the activity of MPX. Four drug treatments, olvanil, dexamethasone, hydrocortisone, and indometbacin, were shown to modulate HD-induced inflammation.

Sulfur mustard (HD) is a bifunctional alkylating agent that has mutagenic, cytotoxic, and vesicating properties (1–3). Although HD preferentially alkylates DNA, it is also known to modify RNA and proteins covalently (4). DNA alkylation has been postulated to be the molecular mechanism that initiates a cascade of events terminating in vesication (5). DNA alkylating agents, like HD, have been shown to cause unbalanced cell growth, a condition that occurs when cells exposed to cytotoxic drugs increase in cell volume, protein, and RNA content as cellular mass accumulates, but the cells fail to undergo cell division (6, 7). Depending on concentration, HD will produce different effects in vitro, ranging from inhibition of proliferation with unbalanced cell growth at lower concentrations to cytotoxicity at higher exposure levels (4,8). With an inhibition of proliferation occurring with HD-exposed human epidermal keratinocytes (HEKs), measures of biochemical parameters taken from cultures at extended times postexposure must be standardized to account for differences in cell number. The primary objective of these studies was to assess the feasibility of using protein as a means of standardizing data across HD treatment groups.

Studies were conducted under this task to Assess the time and concentration dependent nature of niacinamide (NM) protection against HD-induced NAD+ depletion and cytotoxicity. The HD concentrations used assess the time dependent nature of cytotoxicity and NAD+ depletion, and the impact of NM and niacin (NI) treatment were 13,62, 101, and 171 mu-M HD. Three concentrations of NM and NI were (0.01,0.1 and 1 mM) selected by USAMRICD for evaluation at 2, 4,8, 12, 16,20,24,48, and 72 hours after exposure to HD. Cytotoxicity and total culture NAD+ content were assessed. NAD+ concentrations following the addition of 1 mM NM frequently were significantly greater than those observed for the HD-exposed controls especially at 171mu-M HD. Multiple addition of NM had little protective effect relative to that by provided by pretreatment alone. At the 171mu-M HD concentration, the single addition of NM provided marginal but statistically significant (p<=0.05) protection. Comparisons of the different NM addition modes yielded mixed results, but usually the number of viable cells was greater with multiple additions of NM. NI did not provide protection against HD-induced NAD+ depletion or cytotoxicity.

: A study was instituted in sheep to compare efficacy against Soman and the pharmacokinetics of atropine and HI-6 by wet/dry autoinjector and by syringe. The efficacy in sheep against Soman of Atropine/HI-6 delivered by wet/ dry autoinjector was also compared to the efficacy of atropine/pralidoxime chloride when syringe. The aging rate of GD was also determined in sheep erytrocytes. There were no statistical differences in the LD50's of sheep treated with atropine/HI-6 using syringes of wet/dry autoinjectors. A PR was determined for atropine/HI-6 treatment. Based on analyses of HI-6 and atropine pharmacokinetic parameters there were no statistically significant differences in the two injection techniques. The aging rate of sheep RBC's is highly dependent upon the GD-AChE incubation temperature.

A task was instituted at the Medical Research and Evaluation Facility (MREF) to develop in vitro assays to screen pretreatment and treatment compounds for their ability to protect or reverse the toxic effects of organophosphates and vesicants. Four vesicant assays and three nerve agent assays were developed. Two of the vesicant assays were for cell viability of keratinocyte, one in the presence of distilled mustard and one lewisite. One assay determines the effect of vesicants on keratinocyte reproduction and the other the effect of distilled mustard on cellular coenzyme nicotinamide adenine dinucleotide content. The organophosphate assays measure the effects on acetylcholinesterase of selected compounds measured by ability to reactivate, effect on aging rate, and directly. In vitro screen; HD; L; Cellular NAD+ cellular viability; GA; GD; VX; Acetylcholinesterase inhibition; Reactivators; RA 5; Aging rate; Keratinocytes; Treatment and pretreatments; Assaying; Tabun (GA); Sarin (GB); Soman (GD); Organoarsenic; Organophosphates; Chemical Surety Material (CSM); Blisters; Toxicity; Toxic agents; Nerve agents; Chemical warfare agents; G Agents; V Agents; Vesicants; Mustard agents.